Micro-array technology for expression profiling is well known and currently is widely used in genomic research.
Over the past decade much interest has centred on the development of DNA chips based on high density oligonucleotide arrays and fluorescence analysis such as described by Hacia et al. (J. G. Hacia, L. C. Brody, M. S. Chee. S. P. A. Fodor F. S. Collins in Nature Genetics 14, Dec. 1996). One of the examples of commercialisation of this technology has been Affymetrix's “GeneChip”, which was developed to process large amounts of genetic information. Affymetrix technology relies on photolithographic processing to produce thousands of binding sites on a single chip. Alternative techniques include robotic spotting and ink-jet printing although they achieve somewhat smaller binding site density within the micro-array.
For the micro-arrays in common use, one typically starts by taking a specific biological substance or system of interest, extracting its mRNA, and making a fluorescence-tagged cDNA copy of this mRNA. This tagged cDNA copy, typically called the sample probe (sometimes also called target), is then hybridised to a slide containing a grid or array of single stranded cDNA's called probes which have been built or placed in specific locations on this grid. Similar to the general hybridisation principles, a sample probe will only hybridise with its complementary probe i.e. nucleic acid strands tend to be paired to their complements in double-stranded structures. Thus, a single-stranded DNA molecule will seek out its complement in a complex mixture of DNA containing large numbers of other nucleic acid molecules. Hence, nucleic acid probe (e.g., gene probe) detection methods are very specific to DNA sequences. Factors affecting the hybridisation or reassociation of two complementary DNA strands include temperature, contact time, salt concentration, the degree of mismatch between the base pairs, and the length and concentration of the target and probe sequences. In perhaps the simplest procedure, hybridisation is performed on an immobilized probe molecule attached on a solid surface such as a nitrocellulose or nylon membrane or a glass plate.
Fluorescence is typically added to the sample probe in one of two ways: (i) fluorescent nucleotide bases are used when making the cDNA copy of the RNA or (ii) biotinylated nucleotides are first incorporated, followed by an application of fluorescence-labelled streptavidin, which will bind to biotin (I. S. Kohane “Microarrays for integrated genomics” MIT Press, 2002).
Depending on manufacturer specific protocols, the probe-sample probe (probe-target) hybridisation process on a microarray typically occurs over several hours. All unhybridised sample probes are then washed off and the micro-array is lit under laser light and scanned using laser confocal microscopy. A digital image scanner records the brightness level at each grid location on the micro-array corresponding to particular RNA species. The brightness level is correlated with the absolute amount of RNA in the original sample, and by extension, the expression level of the gene associated with this RNA.
Although term hybridisation is typically applied to DNA arrays, in this specification we will use it in a more general sense to also describe binding processes in protein arrays, e.g. binding of antigens to antibodies. It is understood that in protein arrays such binding processes occur over shorter periods of time and at lower temperatures as compared to DNA arrays.
Despite the inherent integration promise, the DNA and protein chips, while, in principle, much like the microprocessor chips that currently run today's computers, have yet to be successfully developed into monolithically integrated single chip devices that conveniently and inexpensively capture, deliver and interpret information that is gathered by what is currently known as “DNA chips” or “biochips”. What is currently understood by term “biochips” is typically a glass slides with an array of binding sites, each site containing specific probe molecules, which requires complex and bulky equipment for external laser excitation, scanning and imaging of the optical signals. In addition to the cost associated with this equipment, there is also a requirement for it to be operated by highly trained and skilled personnel in order to ensure error free interpretation of the gathered data and troubleshooting. These limitations of cost and space associated with the present status of biochip technology currently prevent DNA and protein analysis from finding a wider use in hospitals and eventually in doctor surgeries.
Therefore, there is a distinct requirement for an inexpensive, disposable biochip device that could be interfaced directly to a computer and could be available to any pathology laboratory both in terms of cost and skills required to operate it. Towards that goal it would be further beneficial to simplify the DNA and protein analysis procedure by removing the need for the use of fluorescent markers in detecting specific binding events as well as by removing the washing step after the hybridisation. For research and diagnostic purposes as well as to reduce possible errors in determining a specific binding event it would be also beneficial to enable continuous monitoring of the conditions at each binding site during hybridisation. This latter feature is especially important for the protein arrays due to the relatively unstable nature of protein binding agents (as compared to DNA binding agents).
Over the past few years there has been some effort deployed to reduce cost/size of the biochips by integrating them with the associated laser excitation and image scanning apparatus (Vo-Dinh et al, “Integrated circuit biochip microsystem” U.S. Pat. No. 6,448,064, September 2002; Duveneck et al “Optical detection device based on semiconductor laser array” U.S. Pat. No. 6,469,785, October 2002; Bruno-Raimondi et al “Sensing unit provided with separated detection light guiding” U.S. Pat. No. 6,437,345, August 2002; Neuschafer et al “Sensor platform and method for the parallel detection of a plurality of analytes using evanescently excited luminescence” U.S. Pat. No. 6,078,705, June 2000). These inventions proposed an integrated circuit biochip microsystem, which combines lasers, detectors, focusing optics and biological sensing elements within a single micro-assembly. In microelectronics this type of integration is typically defined as hybrid integration i.e. when individual elements are produced separately by processing a number of separate substrates/wafers and then diced out and micro-assembled together. Although advantageous over the bulky, bench top devices, such hybrid integrated biochips still lack the cost and performance advantage of true monolithic integration. It should be further noted that all these devices require the use of fluorescent markers which unnecessarily complicates the analysis procedure and which ideally should be avoided in a simple point of care devices.
Recently, some work has been done in integration of vertical cavity surface emitting lasers (VCSELs) as light sources and photodetectors on the same III-V semiconductor substrate (GaAs) for fluorescent sensing [E. Thrush et al “Integrated biofluorescence sensor” Journal of Chromatography Vol. A1013, 2003, pp. 103-110]. Although this approach is potentially applicable to manufacturing of individual chemical or biological sensors, it does not offer a suitable technology platform for manufacturing of fully integrated disposable biochips due to inherent cost and substrate size limitations associated with III-V semiconductor compounds.
It is therefore the subject of present invention to propose a monolithically integrated biochip device, as well as a practical and cost effective method of its manufacturing. It is also a subject of the present invention to propose a biochip that can provide a lable-free detection (eliminating the need to use fluorescent markers) and in-situ monitoring of hybridisation conditions at each binding site. Using this invention a remarkable cost/performance ratio reduction can be achieved over the prior art discrete element micro-assembly devices, opening the way for the widespread use of inexpensive, disposable DNA and protein chips.